Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual's body thus proliferate, forming a tumor. When a tumor is found, the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment.
B-raf (or BRAF) encodes a protein that belongs to the Serine/Threonine protein kinases. BRAF is a part of the Ras/Raf/MEK/MAP signal transduction pathway and plays a role in regulating the MAP Kinse/ERK signaling pathway. Mutations in this gene have been associated with various cancers such as colorectal cancer (CRC), non small cell lung cancer (NSCLC), malignant melanomas and adenocarcinomas. Oncogenic mutations in BRAF, nearly all of which are the V600E mutation, have been reported in colon cancer (Davies H, et al. Nature 2002; 417:949-54; Rajagopalan H, et al., Nature 2002; 418:934.). The V600E mutation has been observed in over half of all microsatellite-unstable carcinomas and in a much smaller subset of stable colon tumors (Wang L, et al., Cancer Res 2003; 63:5209-12). The V600E (formerly V599E) mutation is located on exon 15 of the B-raf gene (Accession number NM—04333.4) at position 1860 (1799 of the coding sequence). At position 1799 of the coding sequence, a thymidine is changed to an adenosine, which results in the change from a valine (V) in the wildtype/non mutant B-rag gene to a Glutamine (E) in the mutated gene. In addition, a rare (<1%) V600K (1798-1799 GT>AA) mutation also exists. Furthermore, the V600D mutation exists in 4.6% of cases, the V600A mutation exists in <1% of cases, and the V600M mutation exists in <1% of cases. In addition, there are V600R and K601E BRAF mutations.
The V600E BRAF mutation is found in a number of tissue/tumor types including: nervous system, thyroid, skin, gastrointestinal tract, large intestine, biliary tract, ovary, eye, prostate, central nervous system, liver, small intestine, breast, pancreas, soft tissue, upper, aerodigestive tract, adrenal gland, autonomic ganglia, haematopoietic and lymphoid tissue, lung, esophagus, pituitary, and stomach. DNA or RNA extracted from samples of any of these types of tissues can be utilized in assays of the present invention.
In both stable and unstable cancers, >90% of tumors with BRAF mutations have widespread methylation of CpG islands or what is known as the CpG island methylator phenotype (CIMP). Improved survival associated with microsatellite instability (MSI) in sporadic colon cancers has been reported (Samowitz WS, et al., Cancer Epidemiol Biomarkers Prey 2001; 10:917-23; Halling KC, et al., J Natl Cancer Inst 1999; 91:1295-303), and because sporadic unstable tumors commonly show both CIMP (Toyota M, et al., Proc Natl Acad Sci USA 1999; 96:8681-6; Toyota M, et al., Proc Natl Acad Sci USA 2000; 97:710-5) and BRAF mutations (Kambara T, et al., Gut 2004; 53:1137-44; Nagasaka T, et al., J Clin Oncol 2004; 22:4584-94), one would expect that these features would also show a relationship to improved survival in unstable tumors. Samowitz has studied the relationship between CIMP and survival in microsatellite-stable tumors and has evaluated the relationship between BRAF mutations and survival in microsatellite-stable colon cancers. See Samowitz, Wade S., et al., Cancer Research 65, 6063-6069, Jul. 15, 2005. Samowitz has evaluated a large population-based sample of individuals with colon cancer to determine its relationship to survival and other clinicopathologic variables. The V600E BRAF mutation was seen in 5% of microsatellite-stable tumors and 51.8% of microsatellite-unstable tumors. In microsatellite-stable tumors, this mutation was related to poor survival, CIMP high, advanced American Joint Committee on Cancer (AJCC) stage, and family history of colorectal cancer. The poor survival was observed in a univariate analysis of 5-year survival (16.7% versus 60.0%); in an analysis adjusted for age, stage, and tumor site; in stage-specific, age-adjusted analyses for AJCC stages 2 to 4 (HRR, 4.88, 3.60, and 2.04, respectively); and in Kaplan-Meier survival estimates for AJCC stages 2 to 4. Microsatellite-unstable tumors were associated with an excellent 5-year survival whether the V600E mutation was present or absent (76.2% and 75.0%, respectively). Samowitz has concluded that the BRAF V600E mutation in microsatellite-stable colon cancer is associated with a significantly poorer survival in stages 2 to 4 colon cancer but has no effect on the excellent prognosis of microsatellite-unstable tumors.
Moreover, BRAF mutations proved to be absent in tumors from hereditary nonpolyposis colorectal cancer syndrome (HNPCC) families with germline mutations in the MMR genes MLH1 and MSH2. These data suggest that the oncogenic activation of BRAF is involved only in sporadic colorectal tumorigenesis. The detection of a positive BRAF-V600E mutation in a colorectal cancer suggests a sporadic origin of the disease and the absence of germline alterations of MLH1, MSH2 and also of MSH6. These findings have a potential impact in the genetic testing for HNPCC diagnostics and suggest a potential use of BRAF as exclusion criteria for HNPCC or as a molecular marker of sporadic cancer. See Domingo et al., Oncogene (2005) 24, 3995-3998.
Solit's group have found, using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either ‘wild-type’ cells or cells harboring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both down regulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumor growth in BRAF mutant xenografts, whereas RAS mutant tumors were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumors, and offer a rational therapeutic strategy for this genetically defined tumor subtype. See Solit, David B., et al., Nature 439, 358-362 (19 Jan. 2006).
In addition, a model of human melanocyte transformation has emerged based on the results of genetic studies, cell biology, molecular pathology and mouse modeling. Studies have identified involvement of various factors including basic fibroblast growth factor production, ERK activation, and frequent BRAF mutations in melanoma tissues. BRAF acts downstream of RAS, and studies have demonstrated that simultaneous mutations in RAS and BRAF are extremely rare in melanoma, suggesting that BRAF mutations substitute for at least some of the oncogenic function of mutant RAS.
The development of tumor markers to better stratify patients for their risk of developing metastases is under active investigation. Although assessment of tumor markers and selection of treatment based on the results has been part of the standard of care in colon and breast cancer management for several years, no such markers exist for melanoma. Many studies have shown promise, but none have moved past the preliminary stages of development into a clinically useful assay.
Despite recent advances in the study of melanoma biology, the development of molecular tools useful for diagnosing and/or monitoring patients with melanoma is still relatively new. Few advances have been made in protocols designed to monitor patients for disease recurrence, or to select patients at high risk for the development of metastases. Tumor stage, the best predictor of survival from melanoma, is based on conventional clinicopathologic variables such as thickness and ulceration of the primary tumor, and the presence of metastatic disease in regional lymph nodes or at distant sites. Two patients with primary tumors of intermediate thickness that appear microscopically identical can, however, have dramatically different survivals. The absence of improved prognostic tools for such assessments makes it difficult for attending physicians to determine the best treatment strategies.
Mutations in the BRAF oncogene have been discovered in up to 80% of melanoma tissues, frequencies strikingly higher than any other molecular alteration in this disease. BRAF mutations have also been detected in tumor tissues from other types of cancer. Experimental studies have demonstrated that several BRAF mutations, especially the T1799A (formerly designated T1796A) hotspot mutation, which accounts for 90% of BRAF mutations in melanoma, can transform fibroblasts in culture. Most recently, experiments blocking the expression of mutant BRAF in melanoma cell culture were shown to inhibit cell growth and promote cell death, suggesting that BRAF inhibitors could bolster melanoma treatment significantly.